Combined effect of tumor necrosis factor-alpha and ionizing radiation on the induction of apoptosis in 5637 bladder carcinoma cells.

BACKGROUND AND PURPOSE

Apoptosis can be induced by distinct but overlapping pathways. Ionizing radiation induces apoptosis by an "intrinsic", mitochondria-dependent pathway. Ligation of tumor necrosis factor-(TNF-)alpha, FAS (CD95) or TRAIL receptors are typical representatives of an extrinsic, death-receptor-mediated pathway. In this study the effect of irradiation, treatment with the cytokine TNF-alpha, or a combination of both on the induction of apoptosis and clonogenic survival of bladder carcinoma cells was investigated.

MATERIAL AND METHODS

5637 bladder carcinoma cells were treated with different concentrations of recombinant TNF-alpha (0-10 ng/ml), irradiated with single doses ranging from 0.5 to 10 Gy, or a combination of both modalities. Apoptotic cells were quantified by the TUNEL assay up to 96 h following treatment, clonogenic cell survival by a clonogenic assay. Synergistic effects of both modalities were evaluated using isobolographic analysis.

RESULTS

Irradiation of 5637 carcinoma cells resulted in a discontinuous dose dependence of the apoptotic fraction with a pronounced increase in the range of 0-2 Gy and a slighter increase at 2-10 Gy. The percentage of apoptotic carcinoma cells also increased continuously after treatment with lower concentrations of TNF-alpha reaching a plateau at concentrations of 5.0-10.0 ng/ml. Isobolographic analysis revealed a supraadditive interrelationship between irradiation and TNF-alpha in the range between 0.005 and 0.5 ng/ml, and an additive effect for TNF-alpha concentrations>0.5 ng/ml. The additive effects were confirmed in clonogenic survival assays with reduced survival fractions following combined TNF-alpha administration and irradiation.

CONCLUSION

The combination of two apoptosis-inducing modalities resulted in a synergistic effect on the induction of apoptosis in 5637 bladder carcinoma cells. Although a radiosensitizing effect still has to be proven in animal models, combined-modality treatment may increase the therapeutic effectiveness of irradiation in bladder cancer.